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Heart Stem Cells

Larry H. Bernstein, MD, FCAP, Curator

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Latest in Heart Stem Cell Debate

Given the right environment, cKit+ cells from the mouse heart can develop into new cardiac muscle, according to a study.

By Kerry Grens | October 26, 2015

http://www.the-scientist.com//?articles.view/articleNo/44341/title/Latest-in-Heart-Stem-Cell-Debate/

Cells in the heart expressing the marker cKit were once thought to be the key to cardiac regeneration. These cardiac precursors, researchers found, could proliferate—opening up the opportunity for a way to regrow an organ that until this century was thought incapable of regeneration.

But even as positive results shook out of an early stage clinical trial, a shadow moved in over cKit+ cells, with several labs producing data questioning their reparative powers. Skepticism culminated with a report in 2014 showing that cKit+ cells in mice very rarely produce new heart muscle cells, or cardiomyocytes. The story of cKit+ cells, said Joshua Hare of the University of Miami Miller School of Medicine, “is a very controversial one.”

In the latest development in the cKit+ saga, published this month (October 5) in PNAS, Hare’s team found that cKit+ cells readily become cardiac muscle cells in vitro, as long as the right cellular conditions are present. This could perhaps explain why other groups haven’t seen cKit+ cells becoming cardiomyocytes in vivo that often, he said. “It’s not that the cells don’t have the capacity [to differentiate], but they’re entering the heart at a time that’s nonpermissive for them to become cardiac myocytes.”

Specifically, the researchers found that if they interfered with bone morphogenetic protein signaling—crucial during the development of the heart and other tissues—mouse induced pluripotent stem cells (iPSCs) expressing KIT would become cardiomyocytes. They also demonstrated with genetic fate-mapping that cKit+ cells derive from the neural crest during development and are present in the mouse embryonic heart.

Hare’s group did not find that cKit+ cells have a high propensity to become endothelium, as did the aforementioned 2014 study, which also used genetic fate-mapping. Jeffery Molkentin of Cincinnati Children’s Hospital Medical Center who led that work declined to be interviewed for this story. Hare said the discrepancy could be due to the teams’ different genetic constructs.

Bernardo Nadal-Ginard, an honorary professor at King’s College London whose work has supported the myogenic capacity of cKit+ cells, said he found the evidence from Hare showing they can become myocytes “convincing.” However, he added, “the paper claims the quandary and the dispute is over. But, unfortunately, it is not.”

The paper is more qualitative than quantitative, said Nadal-Ginard, meaning researchers still don’t know how often cKit+ cells become myocytes and whether they become other types of cells (and at what frequency).

Michael Kotlikoff of Cornell University pointed out that Hare’s team didn’t demonstrate that cKit+ cells in vivo have the same regenerative capacity as the iPSCs in vitro. “They never show the myogenic potential of those cells and don’t show them giving rise to cardiomyogensis” in vivo, Kotlikoff told The Scientist. “The expression of [cKit], per se, is not sufficient to identify cells as precursors and the further presumption that signaling processes observed in in vitro differentiation experiments limit such cells from undergoing myogenesis in the adult heart, the stage at which clinical regenerative efforts are focussed, is not supported by data,” he added in an email.

Hare is involved in two planned clinical trials that will administer cKit+ cells to patients with heart failure. (He founded a company called Vestion that is developing cardiac cell therapies.) Already, a phase 1 trial called SCIPIO, which Hare was not part of, found positive signs of tissue repair among patients given their own cKit+ cells. But as questions were raised about the regenerative abilities of these cells, some advocated to wait on the clinical trials until the biology was worked out. Hare said his study does not explain SCIPIO’s results; rather, it offers some clues as to how researchers can boost the reparative potential of these cells.

“To say human trials should be stopped because the experiment didn’t work in the mouse is a bit aggressive,” said Brigham and Women’s Hospital’s Piero Anversa, a leading proponent of cKit+ cells who was involved in SCIPIO and who also found Hare’s results convincing. (Anversa’s own work in the field has been a source of controversy, with an expression of concern issued about some SCIPIO results.) “The answer is going to be in the trial. If the trial goes well we win, if the trial doesn’t go well, we lose.”

K.E. Hatzistergos et al., “cKit⁺ cardiac progenitors of neural crest origin,” PNAS, 112:13051-56, 2015. 

 

More Doubt Cast Over Cardiac Stem Cells

Contrary to previous reports, cell lineage tracing reveals stem cells in the heart rarely contribute to new muscle.

By Kerry Grens | May 7, 2014

http://www.the-scientist.com/?articles.view/articleNo/39912/title/More-Doubt-Cast-Over-Cardiac-Stem-Cells/
FLICKR, GEORGE SHULKINC-kit cells, which are found in the heart and supposedly act as cardiac stem cells, are the basis of a clinical trial to repair cardiac injury. But a new study published in Nature today (May 7) adds what some researchers are calling “definitive” evidence to the idea that these cells hardly ever produce new heart muscle in vivo. Using genetic lineage tracing in a mouse, a team led by Jeff Molkentin of Cincinnati Children’s Hospital Medical Center found that, while c-kit cells readily produce cardiac endothelium, they very rarely generate cardiomyocytes.

“The conclusion I am led to from this is that the c-kit cell is not a cardiac stem cell, at least in term of its normal, in vivo role,” said Charles Murry, a heart regeneration researcher at the University of Washington who was not involved in this study.

The latest findings add to a string of recent setbacks for advancing the use of these cells as a therapy—including a retraction and an expression of concern regarding two publications and an institutional investigation of one of the leaders in the field, Piero Anversa at Harvard Medical School. “There’s been a tidal wave in the last few weeks of rising skepticism,” said Eduardo Marbán, an author of the new study and a cardiologist at the Cedars-Sinai Heart Institute in Los Angeles. Still, he said, the dispute is not settled, and many stand by the regenerative power of these cells.

“Unequivocal” results

Research led by Anversa has shown that c-kit cells—cardiac progenitor cells expressing the cell surface protein c-kit—can produce new cardiomyocytes. Anversa and others have helped usher the cells into clinical trials to test whether they might help repair damaged cardiac tissue.

Work by other teams, however, has raised doubts about the potential for c-kit cells to actually build new heart muscle. To help resolve the discrepancy, Molkentin and his colleagues developed a mouse in which any cell expressing c-kit—and any of that cell’s progeny—would glow green by a green fluorescent protein tagged to the Kit locus. They found that just 0.027 percent of the myocytes in the mouse heart originated from c-kit cells. “C-kit cells in the heart don’t like to make myocytes,” Molkentin told The Scientist. “We’re not saying anything that’s different” from groups that have not had success with c-kit cells in the past, Molkentin said, “we’re just saying we did it in a way that’s unequivocal.”

Molkentin’s study did not address why there’s a discrepancy between his results and those of Anversa and another leader in the c-kit field, Bernardo Nadal-Ginard, an honorary professor at King’s College London. Last year, Nadal-Ginard and his colleagues showed in Cell that heart regeneration in rodents relies on c-kit positive cells and that depleting these cells abolishes the heart’s ability to repair itself. Nadal-Ginard toldThe Scientist that technical issues with Molkentin’s mouse model could have affected his results, causing too few c-kit cells to be labeled. Additionally, “the work presented by Molkentin used none of our experimental approaches; therefore, it is not possible to compare the results,” Nadal-Ginard said in an e-mail.

In an e-mail to The Scientist, Anversa said his lab is working with the same mouse model Molkentin used, “but our data are too preliminary to make any specific comment. Time will tell.”

Clinical future

Molkentin’s paper only serves to darken the cloud that has moved over Anversa’s work on c-kit cells. Last month, a 2012 paper in Circulation by Anversa’s team was retracted because the data were “sufficiently compromised.” Days later, The Lancet published an expression of concern regarding supplemental data in the published results from the human clinical trial using autologous c-kit cells. Harvard Medical School and Brigham and Women’s Hospital continue to investigate what may have gone wrong.

Meanwhile, Marbán is advancing another type of stem cell, called cardiosphere-derived cells, through human clinical trials to try and treat tissue damage after a heart attack. Marbán said he had been a true believer in c-kit cells, until the data started mounting against them. “The totality of the evidence now says the c-kit cell is no longer a cardiomyocyte progenitor,” he told The Scientist.

If c-kit cells don’t produce new cardiomyocytes, as Molkentin and Marbán assert, where does that leave the clinical trial? Murry said that just because the preclinical, mechanistic basis for the human study is foundering, any promising clinical results are not to be dismissed. “Those results can be considered independent,” he said. Molkentin said it’s possible that c-kit cells work in unknown ways to repair heart tissue. He noted that clinical treatment involves high levels of c-kit cells immersed in culture conditions. “Perhaps these cells act a little different,” Molkentin said.

Nadal-Ginard did not dispute that discrepancies exist between his data and those of others, and agreed that these differences ought to be addressed. He said he’d be willing to work with Molkentin to get to the bottom of it. “The concept under dispute is too important for the field of regenerative medicine—and regenerative cardiology, in particular—to turn into a philosophical/dogmatic argument instead of settling it in a proper scientific manner.”

J.H. van Berlo et al., “c-kit1 cells minimally contribute cardiomyocytes to the heart,” Nature, doi:10.1038/nature13309, 2014.

cKit⁺ cardiac progenitors of neural crest origin

Konstantinos E. Hatzistergos^a, Lauro M. Takeuchi^a, Dieter Saur^b, Barbara Seidler^b, Susan M. Dymecki^c, Jia Jia Mai^c, Ian A. White^a, Wayne Balkan^a, Rosemeire M. Kanashiro-Takeuchi^a,^d, Andrew V. Schally^e,¹, and Joshua M. Hare^a,¹

Author Affiliations

Contributed by Andrew V. Schally, August 29, 2015 (sent for review April 27, 2015; reviewed by Roger Joseph Hajjar)

Abstract Full Text Authors & Info Figures Related Content PDF

PNAS Oct 20, 2015; 112(42): 13051-13056 http://dx.doi.org:/10.1073/pnas.1517201112

 

Significance

A high-resolution genetic lineage-tracing study in mice reveals that cKit identifies multipotent progenitors of cardiac neural crest (CNC) origin. Normally, the proportion of cardiomyocytes produced from this lineage is limited, not because of poor differentiation capacity as previously thought, but because of stage-specific changes in the activity of the bone morphogenetic protein pathway. Transient bone morphogenetic protein antagonism efficiently directs mouse iPSCs toward the CNC lineage and, consequently, the generation of cKit⁺ CNCs with full capacity to form cardiomyocytes and other CNC derivatives in vitro. These findings resolve a long-standing controversy regarding the role of cKit in the heart, and are expected to lead to the development of novel stem cell-based therapies for the prevention and treatment of cardiovascular disease.

Abstract

The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches withcKitCreERT2/+ and Wnt1::Flpe mouse lines, we show that cKit delineates cardiac neural crest progenitors (CNCkit). CNCkit possess full cardiomyogenic capacity and contribute to all CNC derivatives, including cardiac conduction system cells. Furthermore, by modeling cardiogenesis in cKitCreERT2-induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNCkit is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit⁺ cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNCkit contribution to myocardium.